uPIC–M: efficient and scalable preparation of clonal single mutant libraries for high-throughput protein biochemistry

Abstract

New high-throughput biochemistry techniques complement selection-based approaches and provide quantitative kinetic and thermodynamic data for thousands of protein variants in parallel. With these advances, library generation rather than data collection has become rate limiting. Unlike pooled selection approaches, high-throughput biochemistry requires mutant libraries in which individual sequences are rationally designed, efficiently recovered, sequence-validated, and separated from one another, but current strategies are unable to produce these libraries at the needed scale and specificity at reasonable cost. Here, we present a scalable, rapid, and inexpensive approach for creating User-designed Physically Isolated Clonal–Mutant (uPIC–M) libraries that utilizes recent advances in oligo synthesis, high-throughput sample preparation, and next-generation sequencing. To demonstrate uPIC–M, we created a scanning mutant library of SpAP, a 541 amino acid alkaline phosphatase, and recovered 94% of desired mutants in a single iteration. uPIC–M uses commonly available equipment and freely downloadable custom software and can produce a 5000 mutant library at 1/3 the cost and 1/5 the time of traditional techniques.

evSeq: Cost-Effective Amplicon Sequencing of Every Variant in a Protein Library

“…In multiplexed NGS, each submitted sample is tagged with a "molecular barcode"-a unique piece of DNA that encodes the sample's original identity-before all samples are sequenced together in the same NGS run. [19][20][21][22][23][24][25] Post sequencing, the barcodes are used to assign individual reads to individual samples. Multiplexed NGS can be outsourced just like Sanger sequencing, and sequencing providers typically charge tens of dollars per sample in a multiplexed sequencing run, yielding on the order of 10 4 -10 5 individual sequences per sample (assuming the run is performed on an Illumina MiSeq instrument).…”

“…Many mutagenesis methods employed in protein engineering (e.g., site-saturation 27 and tile-based mutagenesis 28 strategies) target mutations to a specific position or region in the sequence of a protein, and thus the variants produced can be sequenced with amplicon sequencing to high coverage. 20 Notably, however, even though increasing coverage yields more confident results, it comes with diminishing returns, and it is generally held that coverage in the tens is more than sufficient for effective referencebased identification of mutations (Supplemental Figure S1). 29 Indeed, clinical sequencing of human genomes targets 30x coverage or greater to minimize false base calls.…”